The ETS-1 gene is a member of the ETS-gene family (ETS-1, ETS-2, erg, and elk) and is homologous to the v-ETS oncogene found in the avian leukemia virus, E26. To characterize the product of the ETS-1 gene, we developed a specific monoclonal antibody against a bacterially-expressed v-ETS protein. The ETS-1 gene product in the human T-cell line, CEM, was found to consist of at least six species: four major species with apparent molecular weights of 51 kDa (p5l), 48 kDa (p48), 42 kDa (p42), and 39 kDa (p39); and two minor species of 52 kDa (pp52) and 49 kDa (pp49), that are the phosphorylated forms of p5l and p48, respectively. All of the ETS-1 proteins are related to each other, and are considered products of the ETS-1 gene. Subcellular localization showed that the pp52 and p5l are found mainly in the cytoplasm, while p48 and p39 are found mainly in the nucleus. Specific antibodies against various exons of ETS-1 showed that both p42 and p39 lack a region corresponding to exon VII. Polymerase chain reaction analyses revealed the presence of an additional RNA product that corresponds to mRNA lacking exon VII. These results support our protein data and suggest that the human ETS-1 gene encodes multiple proteins that are generated by at least two distinct mechanisms: alternative splicing of mRNA and protein phosphorylation. The multiple protein isoforms and their heterogeneity of the localization and phosphorylation may imply some functional significance for the ETS-1 gene product.